Effects of Diadenosine Polyphosphates on Tear Secretion in New Zealand White Rabbits

Abstract

Extracellular diadenosine polyphosphates play important signaling functions in a number of physiological responses. Here we show that diadenosine polyphosphates are normal constituents of tear fluid and are potent stimulators of tear secretion through their interaction with P2Y receptors. Diadenosine tetraphosphate (Ap 4 A) and Ap 5 A were found in rabbit tears under basal conditions at concentrations of 2.92 and 0.58 M, respectively. Single applications of UTP, ATP, and Ap 4 A increased tear secretion to 160 Ϯ 8% (n ϭ 16) (P Ͻ 0.001), 131 Ϯ 6% (P Ͻ 0.05), and 162 Ϯ 11% (P Ͻ 0.05) of placebo values, respectively. Ap 4 A, Ap 5 A, and Ap 6 A, but not Ap 2 A and Ap 3 A, were able to stimulate tear secretion in a dose-dependent manner. Concentration-response studies produced pD 2 values of 5.56 Ϯ 0.03, 5.75 Ϯ 0.12, and 5.50 Ϯ 0.09 for Ap 4 A, Ap 5 A, and Ap 6 A, respectively, with Ap 4 A showing the greatest efficacy. Diadenosine polyphosphates also stimulated P2Y 1 and P2Y 2 receptors expressed in 1321N1 cells with no apparent effect on the other P2Y receptors tested. Nonselective P2 antagonists did not modify the tear secretion induced by UTP or Ap 4 A in rabbit eyes in vivo or in cloned receptors, except for a weak but significant reduction in stimulated tear secretion by reactive blue 2. These results suggest that diadenosine polyphosphates stimulate tear secretion via a P2Y receptormediated mechanism. Comparing the effects of diadenosine polyphosphates applied to the rabbit eye and to cloned P2Y receptors, it appears that the P2Y 2 receptor subtype is responsible for the prosecretory effects of these compounds.

Diadenosine tetraphosphate as a potential therapeutic nucleotide to treat glaucoma

“…Ap 4 A has been described as P2Y 1 , P2Y 2 and P2Y 6 selective agonist and P2Y 13 selective antagonist [19][20][21][22]. *** *** Fig.…”

The role of dinucleoside polyphosphates on the ocular surface and other eye structures

“…Diadenosine tetraphosphate have demonstrated their capability to increase the tear volume in the ocular surface (Pintor et al, 2002b). In the case of Ap 4 A the increment is up 60% compared to baseline values in experiments performed with New Zealand white rabbits.…”

“…However, it has not been fully clarified if the effect of these dinucleotides is to induce tear production at the lacrymal gland or in other secretory structures. The studies performed with animal models, suppressing the effect with RB-2, suggest that diadenosine polyphosphates act as agonist of P2Y 2 receptors for increasing the tear production (Murakami et al, 2000;Pintor et al, 2002b). Curiously, the combination of melatonin with Ap 4 A enhances the effect of the diadenosine over the tear secretion in an animal model, but melatonin does not have any effect when it is applied alone.…”

“…The presence of diadenosine polyphosphates in the tear film was studied due to their properties to enhance both tear secretion and to increase the wound healing rate (Guzman-Aranguez et al, 2007, 2011Pintor et al, 2004a;Pintor et al, 2002b). The exogenous addition of these dinucleotides suggested their presence as natural constituents of tears.…”

“…The strips were then processed to be evaluated by High Pressure Liquid Chromatography (HPLC), following the procedure described by Pintor et al (Pintor et al, 1992). The first adenine dinucleotides described in tear were Ap 4 A and Ap 5 A, which were found in New Zealand rabbit tears (Pintor et al, 2002b). Diadenosine tetraphosphate concentration was 2.92 ± 0.28 mM and diadenosine pentaphosphate concentration was 0.587 ± 11 mM.…”